7. SUMMARY. The long-term goal of this proposal is to gain detailed understanding of how the diaphragm ? the main muscle of respiration ? rapidly weakens in response to mechanical unloading, and of the mechanisms whereby the giant elastic protein titin influences this response. The diaphragm is a unique muscle in that it is constantly subjected to mechanical loading. Recent work suggests that diaphragm strength is remarkably sensitive to mechanical unloading, as occurs during mechanical ventilation in the ICU. How unloading affects diaphragm strength is poorly understood. Increasing this understanding is critically important: within hours, diaphragm unloading during mechanical ventilation causes diaphragm weakness in critically ill patients, contributing to weaning failure. The search for the molecular triggers for the development of diaphragm weakness is ongoing. The potential role of mechanosensor proteins, that link unloading to protein turnover, is under-explored but an exciting concept that needs to be studied. A candidate mechanosensor is titin, a giant elastic protein that has been suggested to sense mechanical stress and link this to trophic signalling pathways. This proposal?s aims to understand mechanosensing in the diaphragm in health and disease, and the role of titin therein. Aim 1 will critically test how titin affects muscle trophicity. We will use unilateral diaphragm denervation (UDD). A property that can be observed during UDD is an initial hypertrophy response and we have shown that this hypertrophy of the denervated hemidiaphragm is caused by cyclic passive stretch of diaphragm fibers, and that titin?s elastic properties dictate the magnitude of the response. In this Aim we will identify the titin-based signalling pathways involved. Aim 2 determines the role of titin?s elasticity in PEEP ventilation-induced longitudinal diaphragm atrophy. This work builds on our recent finding that mechanical ventilation with PEEP, which unloads the diaphragm at a shortened length, causes longitudinal atrophy of fibers. Pilot data suggest that titin-based mechanosensing modulates this response. To critically test the role of titin, we will study the effect of PEEP ventilation on longitudinal atrophy in two titin KO mouse models: one with increased titin stiffness and one with lowered. In Aim 3 we will use unique diaphragm biopsies of critically ill patients to validate the findings from aim 1&2 and study whether titin-based mechanosensing contributes to diaphragm weakness. Up/downregulated titin binding proteins will be determined, the significance of which is tested in mouse models through genetic deletion. The innovation of this proposal lies in the novel research foci with innovative guiding hypotheses, its innovative mouse models, unique diaphragm biopsies from mechanically ventilated critically ill patients, and its novel experimental tools. The proposal?s integrative approach is expected to lead to a significant step forward in our understanding of diaphragm function and the role of titin therein.